Barrett's esophagus, the metaplastic change of the distal esophageal epithelium from squamous to columnar, significantly increases the risk of esophageal adenocarcinoma, the prevalent form of esophageal cancer in this country. Understanding the molecular mechanism underlying esophageal epithelial columnar metaplasia is critical to designing molecularly targeted chemopreventative or therapeutic strategies for both Barrett's esophagus and esophageal adenocarcinoma. We propose that reactivation of latent developmental signaling a pathway within the esophagus is a fundamental requirement for Barrett's metaplasia. This project will expand on our previous work that identified Hedgehog (Hh) pathway activation in Barrett's esophagus and demonstrated that forced Hh expression and subsequent downstream Bone morphogenetic protein (BMP) 4 signaling induces columnar changes in esophageal epithelium. We previously identified SOX9, a transcription factor expressed in the embryonic esophagus, as a Hh-BMP4 target gene that is upregulated in Barrett's esophagus and induces expression of columnar genes. We have now identified a second Hh-BMP4 target gene and embryonic esophageal transcription factor, FOXA2, to be expressed in Barrett's esophagus but not in normal adult esophageal epithelium. Our hypothesis is that expression of these two embryonic transcription factors, activated by Hh and BMP4 signaling induced by gastroesophageal reflux disease (GERD) is required for the development of columnar metaplasia. We will attempt to further define the role of Hh and BMP4 signaling in Barrett's esophagus through three specific aims: Aim 1) To delineate how BMP4 upregulates SOX9 and FOXA2 expression in human esophageal epithelial cells; Aim 2) To determine if sequential and combinatorial stable expression of SOX9, FOXA2, and CDX2 in esophageal squamous epithelial cells results in phenotypic features of Barrett's metaplasia; and Aim 3) To determine in vivo expression levels of Hh and BMP4 pathway proteins and their downstream targets (SOX9, FOXA2) and CDX2 in biopsy samples of esophageal squamous epithelium from GERD patients with and without Barrett's esophagus. Through these studies, we expect to define the role Hh-BMP4 signaling plays in establishing columnar metaplasia and determine if there are differences in Hh-BMP4 signaling between GERD patients with and without Barrett's esophagus. These findings would contribute towards our objectives of 1) identifying specific molecular markers that can be used to predict which GERD patients would benefit from aggressive anti-reflux therapies to prevent Barrett's esophagus; 2) designing a molecularly targeted chemopreventative or therapeutic strategy for Barrett's esophagus and esophageal adenocarcinoma; and 3) validating our innovative approach of applying developmental biology to the understanding of Barrett's metaplasia.